DOI QR코드

DOI QR Code

Antibacterial Activity and Probiotic Potential of Lactobacillus plantarum HKN01: A New Insight into the Morphological Changes of Antibacterial Compound-Treated Escherichia coli by Electron Microscopy

  • Sharafi, Hakimeh (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Maleki, Hadi (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Ahmadian, Gholamreza (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Zahiri, Hossein Shahbani (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Sajedinejad, Neda (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Houshmand, Behzad (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB)) ;
  • Vali, Hojatollah (Facility for Electron Microscopy Research, McGill University) ;
  • Noghabi, Kambiz Akbari (Department of Molecular Genetics, National Institute of Genetic Engineering and Biotechnology (NIGEB))
  • Received : 2012.08.02
  • Accepted : 2012.09.24
  • Published : 2013.02.28

Abstract

Among several bacteria examined, an antibacterial-producing Lactobacillus strain with probiotic characteristics was selected and identified based on 16S rRNA gene sequencing. Subsequent purification and mode of action of the antibacterial compounds on target cells including E. coli were investigated. Maximum production of the antibacterial compound was recorded at 18 h incubation at $30^{\circ}C$. Interestingly, antibacterial activity remained unchanged after heating at $121^{\circ}C$ for 45 min, 24 h storage in temperature range of $70^{\circ}C$ to room temperature, and 15 min exposure to UV light, and it was stable in the pH of range 2-10. The active compounds were inactivated by proteolytic enzymes, indicating their proteinaceous nature, and, therefore, referred to as bacteriocin-like inhibitory substances. Isolation and partial purification of the effective agent was done by performing ammonium sulfate precipitation and gel filtration chromatography. The molecular mass of the GFC-purified active compound (~3 kDa) was determined by Tris-Tricine SDS-PAGE. To predict the mechanisms of action, transmission electron microscopy (TEM) analysis of ultrathin sections of E. coli before and after antibacterial treatment was carried out. TEM analysis of antibacterial compounds-treated E. coli demonstrated that the completely altered bacteria appear much darker compared with the less altered bacteria, suggesting a change in the cytoplasmic composition. There were also some membrane-bound convoluted structures visible within the completely altered bacteria, which could be attributed to the response of the E. coli to the treatment with the antibacterial compound. According to the in vivo experiments oral administration of L. plantarum HKN01 resulted in recovery of infected BALB/c mice with Salmonella enterica ser. Typhimurium.

Keywords

References

  1. Aattouri, N., M. Bouras, D. Tome, A. Marcos, and D. Lemonnier. 2001. Oral ingestion of lactic acid bacteria by rats increases lymphocyte proliferation and interferon production. Br. J. Nutr. 87: 367-373.
  2. Aly, E. A. 2007. Characterization of a bacteriocin-like inhibitory substance produced by Lactobacillus plantarum isolated from Egyptian home-made yogurt. Sci. Asia 33: 313-319. https://doi.org/10.2306/scienceasia1513-1874.2007.33.313
  3. Atrih, A., N. Rekhif, A. J. Moir, A. Lebrihi, and G. Lefebvre. 2001. Mode of action, purification and amino acid sequence of plantaricin C19, an anti-Listeria bacteriocin produced by Lactobacillus plantarum C19. Int. J .Food Microbiol. 68: 93-109. https://doi.org/10.1016/S0168-1605(01)00482-2
  4. Blanquet, S., E. Zeijdner, E. Beyssac, J. P. Meunier, S. Denis, R. Havenaar, et al. 2004. A dynamic artificial gastrointestinal system for studying the behavior of orally administered drug dosage forms under various physiological conditions. Phar. Res. 21: 585-591. https://doi.org/10.1023/B:PHAM.0000022404.70478.4b
  5. BSI 1968. Methods on Microbial Examination for Dairy Purposes. British Standards Institution, British Standards House, London.
  6. Casas, I. A. and W. J. Dobrogosz. 2000. Validation of the probiotic concept: Lactobacillus reuteri confers broad spectrum protection against disease in humans and animals. Microbial Ecol. Health Dis. 12: 247-285.
  7. Chang, Y. H., J. K. Kim, H. J. Kim, W. Y. Kim, Y. B. Kim, and Y. H. Park. 2001. Selection of potential probiotic Lactobacillus strain and subsequent in vivo studies. Antonie van Leeuwenhoek 80: 193-199. https://doi.org/10.1023/A:1012213728917
  8. Daeschel, M. A. 1989. Antimicrobial substances from lactic acid bacteria for use as food preservatives. Food Technol. 43: 164-169.
  9. Delves-Broughton, J., P. Blackburn, R. J. Evans, and J. Hugenholtz. 1996. Applications of the bacteriocin nisin. Antonie van Leeuwenhoek. 69: 193-202. https://doi.org/10.1007/BF00399424
  10. De Vuyst, L. and E. J. Vandamme. 1994. Antimicrobial potential of lactic acid bacteria, pp. 91-142. In L. De Vuyst and E. J. Vandamme (eds.). Bacteriocins of Lactic Acid Bacteria: Microbiology, Genetics and Applications. Blackie Academic and Professional, London.
  11. Dobrogosz, W. J. and S. W. Lindgren. 1990. Antagonistic activities of lactic acid bacteria in food and feed fermentations. FEMS Microbiol. Rev. 87: 149-164. https://doi.org/10.1111/j.1574-6968.1990.tb04885.x
  12. Enan, G., A. Essawy, M. Uyttendaele, and J. Debevere. 1996. Antibacterial activity of Lactobacillus plantarum UG1 isolated from dry sausage: Characterization, production and bactericidal action of plantaricin UG1. Int. J. Food Microbiol. 30: 189-215. https://doi.org/10.1016/0168-1605(96)00947-6
  13. FAO/WHO. 2001. Report of a joint FAO/WHO expert consultation on evaluation of health and nutritional properties of probiotics in food including powder milk with live lactic acid bacteria (LAB).
  14. Franz, C. M., M. Du Toit, N. A. Olasupo, U. Schillinger, and W. H. Holzapfel. 1998. Plantaricin D, a bacteriocin produced by Lactobacillus plantarum BFE 905 from ready-to-eat salad. Lett. Appl. Microbiol. 26: 231-235. https://doi.org/10.1046/j.1472-765X.1998.00332.x
  15. Fricourt, B. V., S. F. Barefoot, R. F. Testin, and H. Steven. 1994. Detection and activity of plantaricin F, an antibacterial substance from Lactobacillus plantarum BF001 isolated from processed channel catfish. J. Food Prot. 57: 698-702. https://doi.org/10.4315/0362-028X-57.8.698
  16. Fuller, R. and G. R. Gibson. 1997. Modification of the intestinal flora using probiotics and prebiotics. Scan. J. Gastroenterol. 32: 28-31.
  17. Ghrairi, T., J. Frere, and J. M. Berjeaud. 2005. Lactococcin MMT24, a novel two-peptide bacteriocin produced by Lactococcus lactis isolated from rigouta cheese. Int. J. Food Microbiol. 105: 389-398. https://doi.org/10.1016/j.ijfoodmicro.2005.04.019
  18. Ghrairi, T., J. Frere, J. M. Berjeaud, and M. Manai. 2008. Purification and characterization of bacteriocins produced by Enterococcus faecium from Tunisian rigouta cheese. Food Control 19: 162-169. https://doi.org/10.1016/j.foodcont.2007.03.003
  19. Gilliand, S. E. 1990. Health and nutritional benefits from lactic acid bacteria. FEMS Microbiol. Rev. 87: 175-178. https://doi.org/10.1111/j.1574-6968.1990.tb04887.x
  20. Gonzalez, B., P. Arca, B. Mayo, and J. E. Suarez. 1994. Detection, purification and partial characterization of plantaricin C, a bacteriocin produced by a Lactobacillus plantarum strain of dairy origin. Appl. Environ. Microbiol. 60: 2158-2163.
  21. Hernandez, D., E. Cardell, and V. Zarate. 2005. Antimicrobial activity of lactic acid bacteria isolated from Tenerife cheese: Initial characterization of plantaricin TF711, a bacteriocin-like substance produced by Lactobacillus plantarum TF711. J. Appl. Microbiol. 99: 77-84. https://doi.org/10.1111/j.1365-2672.2005.02576.x
  22. Holo, H., Z. Jeknic, M. Daeschel, S. Stevanovic, and I. F. Nes. 2001. Plantaricin W from Lactobacillus plantarum belongs to a new family of two-peptide lantibiotics. Microbiology 147: 643-651 https://doi.org/10.1099/00221287-147-3-643
  23. Jimenez-Diaz, R., R. M. Rios-Sanchez, M. Desmazeaud, J. L. Ruitz-Barba, and J. C. Piard. 1993. Plantaricins S and T, two new bacteriocins produced by Lactobacillus plantarum LPCO10 isolated from a green olive fermentation. Appl. Environ. Microbiol. 59: 1416-1424.
  24. Kelly, W., R. V. Asmundson, and C. M. Huang. 1996. Characterization of plantarum KW30, a bacteriocin produced by Lactobacillus plantarum. J. Appl. Bacteriol. 81: 657-662.
  25. Klaenhammer, T. R. 1993. Genetics of bacteriocins produced by lactic acid bacteria. FEMS Microbiol. Rev. 12: 39-85. https://doi.org/10.1111/j.1574-6976.1993.tb00012.x
  26. Ko, S. H. and C. Ahn. 2000. Bacteriocin production by Lactococcus lactis KCA2386 isolated from white kimchi. Food Sci. Biotechnol. 9: 263-269.
  27. Maidak, B., J. R. Cole, C. T. Parker, G. M. Garrity, N. Larsen,B. Li, et al. 1999. A new version of the RDP (Ribosomal Database Project). Nucl. Acids Res. 27: 171-173. https://doi.org/10.1093/nar/27.1.171
  28. Marteau, P., M. Minekus, R. Havenaar, and J. H. J. Huis In't Veld. 1997. Survival of lactic acid bacteria in a dynamic model of the stomach and small intestine: Validation and the effects of bile. J. Dairy Sci. 80: 1031-1037. https://doi.org/10.3168/jds.S0022-0302(97)76027-2
  29. Medici, M., C. G. Vinderola, R. Weill, and G. Perdigón. 2005. Effect of fermented milk containing probiotic bacteria in the prevention of enteroinvasive Escherichia coli infection in mice. J. Dairy Res. 72: 243-249. https://doi.org/10.1017/S0022029905000750
  30. Messi, P., M. Bondi, C. Sabia, R. Battini, and G. Manicardi. 2001. Detection and preliminary characterization of a bacteriocin (plantaricin 35d) produced by a Lactobacillus plantarum strain. Int. J. Food Microbiol. 64: 193-198. https://doi.org/10.1016/S0168-1605(00)00419-0
  31. Nettles, C. G. and S. F. Barefoot. 1993. Biochemical and genetic characteristics of bacteriocins of food-associated lactic acid bacteria. J. Food Prot. 56: 338-356. https://doi.org/10.4315/0362-028X-56.4.338
  32. Ogunbanwo, S. T., A. I. Sanni, and A. A. Onilude. 2003. Characterization of bacteriocin produced by Lactobacillus plantarum F and Lactobacillus brevis OG1. Afr. J. Biotechnol. 2: 219-227. https://doi.org/10.5897/AJB2003.000-1045
  33. Oh, S., S. H. Kim, and R. W. Worobo. 2000. Characterization and purification of a bacteriocin produced by a potential probiotic culture, Lactobacillus acidophilus 30SC. J. Dairy Sci. 83: 2747-2752. https://doi.org/10.3168/jds.S0022-0302(00)75169-1
  34. Oyetayo, V. O. and B. Osho. 2004. Assessment of probiotic properties of a strain of Lactobacillus plantarum isolated from fermenting corn slurry. J. Food Agric. Environ. 2: 132-134.
  35. Ponce, A. G., M. R. Moreira, C. E. Valle, and S. I. Roura. 2008. Preliminary characterization of bacteriocin-like substances from lactic acid bacteria isolated from organic leafy vegetables. LWT Food Sci. Technol. 41: 432-441. https://doi.org/10.1016/j.lwt.2007.03.021
  36. Powell, J. E., R. C. Witthuhn, S. D. Todorov, and L. M. T. Dicks. 2007. Characterization of bacteriocin ST8KF produced by a kefir isolate Lactobacillus plantarum ST8KF. Int. Dairy J. 17: 190-198. https://doi.org/10.1016/j.idairyj.2006.02.012
  37. Rekhif, N., A. Atrih, and G. Lefebvre. 1994. Characterization and partial purification of plantaricin LC74, a bacteriocin produced by Lactobacillus plantarum LC74. Biotechnol. Lett. 16: 771-776. https://doi.org/10.1007/BF00133952
  38. Rekhif, N., A. Atrih, and G. Lefebvre. 1995. Activity of plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6 isolated from fermented sausages. J. Appl. Bacteriol. 78: 349-358. https://doi.org/10.1111/j.1365-2672.1995.tb03417.x
  39. Rojo-Bezares, B., Y. Saenz, L. Navarro, M. Zarazaga, F. Ruiz- Larrea, and C. Torres. 2007. Coculture-inducible bacteriocin activity of Lactobacillus plantarum strain J23 isolated from grape must. Food Microbiol. 24: 482-491. https://doi.org/10.1016/j.fm.2006.09.003
  40. Sainte-Marie, G. 1962. A paraffin embedding technique for studies employing immunofluorescence. J. Histochem. Cytochem. 10: 250-256. https://doi.org/10.1177/10.3.250
  41. Schillinger, U. and F. K. Lucke. 1987. Identification of lactobacilli from meat and meat products. Food Microbiol. 4: 199-208. https://doi.org/10.1016/0740-0020(87)90002-5
  42. Stevens, K. A., B. W. Sheldon, N. A. Klapes, and T. R. Klaenhammer. 1991. Nisin treatment for inactivation of Salmonella species and other Gram-negative bacteria. Appl. Environ. Microbiol. 57: 3613-3615.
  43. Ten-Brink, B., M. Minekus, J. M. Van-der Vossen, R. J. Leer, and J. H. Huis in't-Veld. 1994. Antimicrobial activity of lactobacilli: Preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus M46. J. Appl. Bacteriol. 77: 140-148 https://doi.org/10.1111/j.1365-2672.1994.tb03057.x
  44. Todorov, S. D., H. Nyati, and M. Meincken. 2007. Partial characterization of bacteriocin AMA-K, produced by Lactobacillus plantarum AMA-K isolated from naturally fermented milk from Zimbabwe. Food Control 18: 656-664. https://doi.org/10.1016/j.foodcont.2006.03.003
  45. Todorov, S. D. and L. M. Dicks. 2005. Lactobacillus plantarum isolated from molasses produces bacteriocins active against Gram-negative bacteria. Enzyme Microb. Technol. 36: 318-326. https://doi.org/10.1016/j.enzmictec.2004.09.009
  46. Vander Merwe, I. R., R. Bauer, T. J. Britz, and L. M. Dicks. 2004. Characterization of thoeniicin 447, a bacteriocin isolated from Propionibacterium thoenii strain 447. Int. J. Food Microbiol. 92: 153-160. https://doi.org/10.1016/j.ijfoodmicro.2003.09.004
  47. Van Reenen, C. A., L. M. T. Dicks, and M. L. Chikindas. 1998. Isolation, purification and partial characterization of plantaricin 423, a bacteriocin produced by Lactobacillus plantarum. J. Appl. Microbiol. 84: 1131-1137. https://doi.org/10.1046/j.1365-2672.1998.00451.x
  48. Vignolo, G., S. Fadda, M. N. De-Kairuz, A. A. de-Ruiz Holgado, and G. Oliver. 1996. Control of Listeria monocytogenes in ground beef by Lactocin 705; a bacteriocin produced by Lactobacillus casei CRL 705. Int. J. Food Microbiol. 29: 397-402. https://doi.org/10.1016/0168-1605(95)00038-0
  49. Vizoso-Pinto, M. G., C. M. A. P. Franz, U. Schillinger, and W. Holzapfel. 2006. Lactobacillus spp. with in vitro probiotic properties from human faeces and traditional fermented products. Int. J. Food Microbiol. 10: 205-214.
  50. Zamfir, M., R. Callewaert, and P. Cornea. 1999. Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801. J. Appl. Microbiol. 87: 923-931. https://doi.org/10.1046/j.1365-2672.1999.00950.x

Cited by

  1. Anti-Infective Activities of Lactobacillus Strains in the Human Intestinal Microbiota: from Probiotics to Gastrointestinal Anti-Infectious Biotherapeutic Agents vol.27, pp.2, 2013, https://doi.org/10.1128/cmr.00080-13
  2. Lactobacillus crustorum KH: Novel Prospective Probiotic Strain Isolated from Iranian Traditional Dairy Products vol.175, pp.4, 2013, https://doi.org/10.1007/s12010-014-1404-2
  3. Escherichia coli-Derived Uracil Increases the Antibacterial Activity and Growth Rate of Lactobacillus plantarum vol.26, pp.5, 2016, https://doi.org/10.4014/jmb.1601.01063
  4. Characterization of bacteriocins produced by strains of Pediococcus pentosaceus isolated from Minas cheese vol.68, pp.6, 2013, https://doi.org/10.1007/s13213-018-1345-z
  5. Bioprospecting Antimicrobials from Lactiplantibacillus plantarum: Key Factors Underlying Its Probiotic Action vol.22, pp.21, 2013, https://doi.org/10.3390/ijms222112076